Ben Forbes - All IPM Flashcards
Limitations of Preservative Efficacy Testing
The 4 organisms (or the organisms) inoculated with sample are not representative of all possible contaminants Lab strains of bacteria have different patterns of resistance to the wild stains contaminating the product during use or manufacture.
28 days cannot be extrapolated to an expected shelf life of 2-3 years (which is the shelf life of API) so will the preservative function for this long time period? Sample of 1 ml will be unable to detect <1 organism/ml
Only a certain type of product needs Preservative efficacy testing name two types
Oral mixtures (to check for absence of E.Coli) and high in sugar products, to check for absence of Saccharomyces
What is a Preservative Efficacy Test?
a 28 day est to verify whether or not the preservative strategy is effective for the product
How do hazards occur?
Hazards occur by the product spoiling due to:
- Breakdown of the active agent by microbes (e.g Atropine eye-drops degraded from pseudomonas)
- Breakdown of the formulation (e.g polysaccharides)
- Reduced acceptability (due to microbes waste)
- Degredation of preservative (e.g phenol -> Pseudomonas
Overally an unfit for use by patient
What parameters define Microbial Contamination?
Hazards (product spoilage)
Limits (the design of ‘sterile’ and non-sterile priducts)
Control measures (GMP, Formulation and Preservatives)
Formulation factors affecting Microbial Content
- Water content: Suspensions, aqueous meds are most susceptible
- Nutritional value
E.g. aromatic water such as peppermint have short T1/2
Pseudomonas can survive in distilled water with limited nutrition available
- pH - most bacteria optimal ~ 7
- Osmotic pressure: Decreases growth
- Surface tension
- Oxygen tension: Store products under Nitrogen to inhibit aerobic microbes
- Storage
- Temp
What tool do we use to define the paramateters of microbial contamination?
In-house guideleines for hospitals and industry, and the BP:
Pharmacopeia
>states the regulatory controls
- Works within limits
- Total viable counts changes for FORM of meds
- Oral, inhalation
- Herbal meds have higher limits than synthetic meds
- Absence of specific organism
- S. auerus and pseudomonas affects inhalation products
- If microbe present = product fails
what tests are there for Microbial Quality Assurance?
- Test for microbial contamination
- Preservative efficacy test
- Sterility test – a count test, where the no. (Of microbes) is 0, so not too different to test for microbial contamination.
How does the Test for Microbial Contamination work?
Work towards the BP limits and European pharmacopeia limits. The route of administration determines what limits permitted and not permitted.
E.g. things for the skin (topical applications) must be free from opportunistic skin pathogens. So things such as S.aureus and pseudomonas and for oral preparation, such as tablets, E.coli
Principles of viable counting techniques
Generally bacteria are faster growing than fungi
- So the bacteria have a different type of agar that they grow in compared to fungi
- For BACTERIA: Casein soya bean digest agar
- For FUNGI: Sabouraud Glucose Agar with Abx [to stop bacteria growing]
How do i do a plate count in “viable counteng techniques”?
50-300 cfu/ml
- 1ml sample added to molten agar OR spread onto the surface of an over dried agar plate.
- For BACTERIA: Casein soya bean digest agar- bacteria (incubate 30-35 °C – their optimum temperature for 5 days)
- For FUNGI: Sabouraud Glucose Agar with Abx [to stop bacteria growing] (incubate 20 -25 °C for 5 days)
Discuss how to do membrane filtration for testing the presence of microbes
For 10-100 cfu/ml
The organisms need to be retained on the surface of a filter.
- 10ml sample filtered through 0.45µm pore size filter
- Filter washed with 3 x 100ml buffer -> to ensure no false results
- Filter transferred to agar plate to encourage growth
what is a serial dilution? what is it based on, and what are the pros.
its a form of viable counting. Based on most probable number (MPN). It is a statically based assay
- A series of dilutions are made and each dilution is inoculated into several tubes of broth
- Incubate 30 - 35°C for 5 days , then observe for growth OR no growth.
- Reference to tables -> to estimate MPN
- Good method if # of microbes is unknown
- Good for a wide range of microbes
How to detect specific organisms
Why do we have preservatives
Protection against:
- Residual contamination not removed by GMP in non-sterile product
- Contamination introduced during use
e.g. creams, eye drops
when selecting a preservative, what main factors do we consider?
- Type of formulation
- Ingredients
- Pysico-chemical properties
- Route of admin
- Non-irritant
- Non-sensitizing
- Non- toxic
- Colourless
- No taste
- Inexpensive
Name examples of preservative and what pH they are effective at
Benzoic and Scorbic acid as a pka of 4.2-5, and are active at lower pH’s
Phenolics are only affective above pH 9
Cationic preservatives are inactive below pH 4-5 e.g Chlorhexidine
Any solubility issues with preservatives? Examples?
Solubilisation (micellar systems)
- Preservative is solubilised above the critical micelle conc (CMC) and is not solubilised below this.
- preservatives will favourable partition into micelle as they are lipophilic, out of the aq phase.
- but the microbes prefer the aqueous phase
X causes reduction in preservative in aqueous phase, where bugs live!
Example: Parabens preservative
Explain this equation
equation showing Effect of dilution on preservative
- t1 and t2 = time required to reduce a viable population to the same extent using the preservative at different concentration; C1 and C2
- indicates reduction in preservative activity due to dilution
- e.g. Phenol activity = 6, when diluted 4-foldà activity reduces by 46 = 4096 times
Why is preservative solubility important and how can we increase preservative solubility?
Preservatives must be soluble in aq phase of a formulation to ensure antimicrobial activity.
Co-solvents can increase solubility AND activity, in the order of:
Ethanol > propylene glycol > glycerol
Explain how the light blockage method works
A high intensity light source is used shone on the particle as it passes through the detection chamber. The particle passes through the light source (typically a laser or halogen light) and scatters the light which is detected by a photo detector.
Detects
Particles > 2um:
Particles > 5um:
